Factors Influencing Genetic Transformation and Plant Regeneration of Vitis

Factors Influencing Genetic Transformation and Plant Regeneration of Vitis Sadanand A. Dhekney, 1 Zhijian T. Li, 1 Thomas W. Zimmerman, 2 and Dennis J. Gray 1 * Abstract: The effect of callus induction media, culture protocols, embryogenic culture age and cotyledon excision treatment on the production of transgenic embryo and plant lines of Vitis was studied. Embryogenic cultures initiated from leaves or stamens and pistils were transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptii) fusion gene. The production of transgenic embryo lines on different culture media was genotype-dependent. Vitis vinifera produced the most transgenic embryo lines (7.5 to 26%) when cultured on DM and X6 medium, whereas Vitis champinii and certain Vitis hybrids produced the best response on NB and X6 medium (5 to 12.5%). Among Vitis vinifera genotypes, Merlot, Superior Seedless, and Thompson Seedless produced transgenic embryo lines irrespective of 4, 8, or 12 month culture ages (11.6 to 36%), whereas Cabernet franc, Cabernet Sauvignon, and Shiraz produced embryo lines only from 4 month cultures (4.1 to 16%). The effect of cotyledon excision of germinated embryos on recovery of transgenic plants varied with variety. The treatment resulted in better plant recovery in Vitis vinifera and Vitis riparia (26.7 to 73.3%), whereas it resulted in a lower plant recovery in Vitis champinii and Vitis hybrids. Transgenic plants were recovered from 19 Vitis genotypes comprised of the species Vitis champinii, Vitis riparia, Vitis rupestris, Vitis vinifera, and Vitis interspecific hybrids. PCR and quantitative real-time PCR confirmed the presence and copy number of the egfp transgene in selected transgenic plants. Transgenic vines established in the field exhibited normal vegetative and reproductive growth compared to non-transformed vines. Stable GFP (green fluorescence protein) expression patterns in mature plant parts of transgenic plants have been observed for over 4 years indicating no transgene silencing. Key words: grapevine, Agrobacterium, somatic embryos, transgenic plants, EGFP Genetic improvement of Vitis has been accomplished by clonal selection of spontaneous bud mutations and breeding (Olmo 1942, Einset and Pratt 1975). However, the occurrence of bud mutations is random, which limits directed crop improvement. Breeding has limited application for genetic improvement because of extreme heterozygosity of the Vitis genome, which is fostered by inbreeding depression (Winkler et al. 1974). Inbreeding depression makes backcrossing and recurrent selection difficult. In particular, for varieties used in wine production where the enological characteristics are finely appreciated, it is impossible to introduce a useful trait via breeding without disrupting the desired phenotype. In addition, the long juvenile period 1 Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL 32703; and 2 Biotechnology & Agroforestry, University of the Virgin Islands, RR 1, Box 10,000, Kingshill, St. Croix, VI 00850. *Corresponding author (email: djg@ufl.edu; tel: 407 884-2034; fax: 407 814-6186) Acknowledgments: This research was supported by the USDA Tropical & Subtropical Agriculture Research Program, the Florida Department of Agriculture and Consumer Services Viticulture Trust Fund, and the Florida Agricultural Experiment Station. The authors thank Seenivasan Natarajan for assistance with statistical analysis. Manuscript submitted Sep 2008, revised Jan 2009, accepted Mar 2009 Copyright 2009 by the American Society for Enology and Viticulture. All rights reserved. of vines makes screening of new selections tedious and time-consuming (Alleweldt and Possingham 1988). Genetic transformation offers a potential alternative for adding single traits, such as disease resistance, to elite varieties without changing desirable characteristics (Gray et al. 2005). Genetic transformation has been the subject of intense study for the past two decades (reviewed by Gray et al. 2005). Transgenic grapevines were obtained using both particle bombardment (Gray et al. 1993) and Agrobacteriummediated transformation; however, the latter was used more widely and with greater success (Das et al. 2002, Hoshino et al. 1998, Iocco et al. 2001, Nakano et al. 1994, Perl et al. 1996, Scorza et al. 1996, Xue et al. 1999, Yamamoto et al. 2000). Despite efforts to genetically engineer grapevine, relatively few varieties were successfully transformed and transformation efficiencies were low. Significant limitations encountered include poor embryogenic potential of genotypes, wide variations among varieties in their response to genetic transformation, Agrobacterium-induced postcocultivation necrosis of embryogenic cultures, and poor plant recovery from transformed somatic embryos (Iocco et al. 2001, Perl et al. 1996). We recently reported an optimized procedure for genetic transformation of Vitis vinifera Thompson Seedless and recovered a high number of independent transgenic plant lines (Li et al. 2006). The optimized protocol was further improved to reduce Agrobacterium-induced postcocultivation necrosis, increase transient and stable gene 285

286 Dhekney et al. expression, and subsequent plant recovery from transgenic somatic embryos in V. vinifera L. and Vitis rotundifolia Michx. (Li et al. 2008, Dhekney et al. 2008). In the current study, we evaluate the improved protocol for its applicability to a range of Vitis species and interspecific hybrids. Additionally, the effects of embryogenic culture age, callus induction media, and cotyledon excision treatment on recovery of transgenic embryo and plant lines was studied in Vitis champinii Planch., Vitis riparia Michx., Vitis rupestris Scheele., V. vinifera, and Vitis interspecific hybrids. Transgenic plants were produced from 19 genotypes in this study, marking a significant improvement in genetic transformation technology. The optimization of parameters influencing transformation, as reported herein, will facilitate rapid testing of candidate genetic elements in Vitis to improve elite genotypes. Materials and Methods Culture initiation and maintenance. Embryogenic cultures of V. champinii Ramsey, V. riparia Gloire, V. rupestris St. George, V. vinifera Cabernet franc, Cabernet Sauvignon, Chardonnay, Merlot, Orange Muscat, Pinot noir, Sauvignon blanc, Shiraz, and Zinfandel, and Vitis hybrids Conquistador, Freedom, Harmony, and Richter 110 were initiated from stamens and pistils (Gray and Mortensen 1987), whereas Superior Seedless, Thompson Seedless, and Seyval blanc cultures were initiated from leaves (Gray 1995) as previously described. After initiation, cultures were transferred to X6 medium consisting of a modified MS medium lacking glycine and supplemented with 3.033 g L -1 KNO 3 and 0.364 g L -1 NH 4 Cl, 60.0 g L -1 sucrose, 1.0 g L -1 myo-inositol, 7.0 g L -1 TC agar (catalog # A 175; Phytotechnology Laboratories, Shawnee Mission, KS), 0.5 g L -1 activated charcoal, and ph adjusted to 5.8 prior to autoclaving (Dhekney et al. 2008, Li et al. 2008). Somatic embryo (SE) development and proliferation occurred on X6 medium. Cultures were maintained by careful transfer of proembryonic masses (PEM) to fresh medium at 4 to 6 week intervals. Transformation vector, bacterial strain. An egfp/ nptii fusion gene under the control of a double Cassava Vein Mosaic Virus (CsVMV) promoter was cloned into the T-DNA region of a pbin19 derived binary vector (Li et al. 2001) and introduced into Agrobacterium tumefaciens strain EHA 105. The bacterial culture was grown as described previously (Li et al. 2006). Briefly, bacteria were cultured overnight in MG/L medium containing 20 mg L -1 rifampicin and 50 mg L -1 kanamycin on a rotary shaker at 180 rpm. The cells were pelleted by centrifugation at 6000 rpm for 8 min, and the pellet was transferred to 25 ml X2 medium for an additional 4 hr of culture before being used to inoculate embryogenic cultures. Transformation of embryogenic cultures. Somatic embryos at the mid-cotyledonary stage of development were used in transformation experiments. SE were cocultivated with Agrobacterium in the dark for 72 hr as previously described (Li et al. 2008), cultured for 3 days in 125-mL conical flasks containing 30 ml liquid callus induction medium (described below) with carbenicillin and cefotaxime (200 mg L -1 each) and then transferred to solid callus induction medium. Callus induction media, recovery of transgenic SE lines. Three distinct callus induction media and protocols DM (Li et al. 2001), GS1CA (Franks et al. 1998), and NB (Le Gall et al. 1994) were compared to study their effect on transgenic callus formation and recovery of transgenic SE lines. DM medium consisted of DKW basal salts (Driver and Kuniyuki 1984), 0.3 g L -1 KNO 3, 1.0 g L -1 myo-inositol, 2.0 mg L -1 each of thiamine-hcl and glycine, 1.0 mg L -1 nicotinic acid, 30 g L -1 sucrose, 5.0 µm benzyladenine (BA), 2.5 µm each β-naphthoxyacetic acid (NOA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and 7.0 g L -1 TC agar at ph 5.7. GS1CA medium was composed of Nitsch and Nitsch (1969) basal salts, Fe-EDTA (Murashige and Skoog 1962), 60 g L -1 sucrose, 10 µm NOA, 1 µm BA, 20 µm indole acetic acid (IAA), 10 g L -1 bactoagar, and 0.25 g activated charcoal at ph 6.2. NB medium consisted of half-strength MS basal salts and vitamins (Murashige and Skoog 1962), 30 g L -1 sucrose, 5 µm NOA, 2.5 µm BA, 0.1g L -1 myo-inositol, and 7.0 g L -1 TC agar at ph 5.8. Somatic embryo explants of Cabernet franc, Merlot, Superior Seedless, Thompson Seedless, Harmony, Richter 110, Ramsey, and Seyval blanc were cocultivated with Agrobacterium and then cultured in 125-mL conical flasks containing 30 ml liquid DM, GS1CA, or NB medium with 200 mg L -1 each of carbenicillin and cefotaxime for 3 days. SE were then transferred to 100 x 15 mm Petri dishes containing 25 ml solid induction medium. SE from liquid DM medium were transferred to solid DMcck medium (DM medium containing 200 mg L -1 each of carbenicillin and cefotaxime, and 100 mg L -1 kanamycin) for 4 weeks. Transgenic calli from DMcck medium were transferred to Petri dishes containing 30 ml X6cck70 medium for development of SE lines. SE explants from liquid GS1CA medium were transferred to solid GS1CA medium with 1 g L -1 timentin and cultured for 3 weeks. Cultures were then grown on GS1CA medium with 100 mg L -1 kanamycin and 1 g L -1 timentin for 5 weeks in the dark followed by transfer to growth-regulator-free GS1CA medium with the same amount kanamycin and timentin as above for recovery of transgenic SE lines. SE explants washed in liquid NB medium were cultured on solid NBcck medium (NB medium containing 200 mg L -1 each of carbenicillin and cefotaxime, and 100 mg L -1 kanamycin) for 90 days and then transferred to X6cck70 medium for secondary embryo development from transgenic callus. Each treatment consisted of 30 SE explants, replicated four times (i.e., 120 SE explants per treatment). The number of transgenic calli and SE lines obtained from each medium were determined based on GFP (green fluorescence protein) and resistance to kanamycin in the culture medium. Experiments were repeated three times. Embryogenic culture age, transgenic callus, SE formation. The effect of embryogenic culture age on recovery

Genetic Transformation of Vitis 287 of transgenic callus and SE lines was studied using Cabernet franc, Cabernet Sauvignon, Merlot, Shiraz, Seyval blanc, Superior Seedless, and Thompson Seedless cultures at 4, 8, and 12 months (mo) after initiation. SE explants were cocultivated with Agrobacterium as described above. Following cocultivation and washing, explants were transferred to DMcck medium and grown in the dark for 30 days. Each treatment consisted of 30 SE explants in a 100 x 15 mm Petri dish containing 25 ml DMcck medium, which was replicated four times. Transgenic calli produced on DM medium were transferred to X6cck70 medium for SE development. Cultures were transferred to fresh medium at 6 week intervals for up to 2 years. The number of transgenic callus and SE lines obtained from each treatment was recorded. SE germination and plant recovery. Transgenic SE at the late cotyledonary stage were germinated in 100 x 15 mm Petri dishes containing 25 ml MS1B medium (Dhekney et al. 2008). Cultures were incubated at 25 C with an 18-hr cool white fluorescent light (60 µmole m -2 s -1 ) / 6-hr dark cycle. Germinated SE became greatly enlarged and pigmented. In particular, cotyledons turned green in color and enlarged during 2 weeks of culture as previously described (Li et al. 2008). To evaluate whether the presence of cotyledons influenced shoot development, they were excised from germinated SE. The control consisted of SE in which the cotyledons were not excised. The time required for shoot development from germinated SE and percent plant recovery per SE was recorded. Resulting plants consisting of a shoot and root system were cultured in Magenta GA7 vessels containing 25 ml MS medium for 3 weeks. Plants were then transferred to 7-cm plastic pots containing Pro-Mix BX potting mix (Premier Horticulture, Red Hill, PA) and acclimated in a growth room for 2 weeks before transfer to a greenhouse. Treatments compared the effect of cotyledon excision on plant recovery for V. vinifera Cabernet franc, Cabernet Sauvignon, Orange Muscat, Superior Seedless, V. champinii Ramsey, V. riparia Gloire, and Vitis hybrids Harmony and Richter 110. Each treatment was replicated four times, a replicate consisting of 5 SE per Petri dish. Molecular analysis of transgenic plants. Polymerase chain reaction and quantitative real-time PCR was used to confirm the presence and copy number of the egfp transgene in transgenic plants. Genomic DNA was extracted from leaves of transgenic plants of V. champinii Ramsey, V. riparia Gloire, V. vinifera Merlot, Vitis hybrid Harmony, and a non-transgenic V. vinifera plant as described previously (Li et al. 2006). The presence of the egfp transgene in transgenic plant lines was detected using PCR. A forward primer EG-51 (5 -ATGGTGAGCAAGGGCGAG- GAGCTGT-3 ) and a reverse primer EG-32 (5 -CTTGTA- CAGCTCGTCCATGCCGAGA-3 ) were used to amplify a 717 bp DNA fragment from the egfp/nptii fusion gene. Conditions for PCR reactions were: 1 cycle at 95 C for 4 min, 39 cycles at 94 C for 1 min, 58 C for 1 min, 72 C for 1 min, and a final cycle at 72 C for 4 min. Plasmid DNA used in transformation served as a positive control whereas DNA from a non-transformed V. vinifera plant was used as a negative control. Transgene copy number in transgenic plants was determined using quantitative real-time PCR as previously described (Dutt et al. 2008). Quantitative real-time PCR assays were carried out in a LightCycler 480 instrument equipped with a 96-well plate Therma-base and software release v1.5 (Roche Molecular Biochemicals, Indianapolis, ID). Oligonucleotide primers for amplification of a 340 bp target fragment from the EGFP gene included a forward primer ERT-51 (5 -CCATCCTGGTCGAGCTGGAC-3 ) and a reverse primer ERT-32 (5 -TTCAGCTCGATGCG- GTTCAC-3 ). All reactions were carried out in a 20 µl final volume containing 2 µl sample DNA (total of 6 ng), 10 µl 2x SYBR Green I Master PCR buffer (catalog # 04707516001; Roche), 2 µl of each primer (0.5 µm), and 4 µl sterile water. Samples were replicated three times for each run and experiments were repeated twice. External controls representing 1 5 copies of the EGFP gene were prepared by diluting EcoRI-linearized EGFP gene-containing plasmid pu203 to a given concentration and adding the DNA to reaction mixtures that contained 6 ng non-transformed grape DNA (Thompson Seedless). The amount of plasmid DNA corresponding to a single copy of the EGFP gene per reaction was calculated as 6 ng (genomic DNA amount) x 5.352*10e3 bp (size of pu203)/4.75*10e8 bp (1C genome size of grape) = 0.0676 pg. Quantitative real-time PCR conditions were as follows: 95 C for 10 min followed by 45 thermal cycles of 95 C for 5 sec, 56 C for 10 sec, and 72 C for 40 sec, with a ramping rate of 4.4 C/sec, 2.2 C/ sec, and 4.4 C/sec, respectively. The level of SYBR-specific fluorescence (483 533 nm) at the end of each cycle was measured and recorded via a CCD camera connected to the LightCycler 480 and further analyzed using the software. Following the thermal cycling, a melting curve analysis was performed to verify sequence specificity of amplified products. Analysis consisted of heating and incubating the sample plate at 95 C for 5 sec at a ramp rate of 4.4 C/sec, lowering the temperature to 50 C (with ramp rate at 2.2 C/ sec) for 1 min, followed by heating the sample plate to a 95 C with a ramp rate of 0.11 C/sec. Fluorescence change profiles in relation to thermal dynamic dissociation of DNA were recorded during the final heating step via continuous data acquisition process (5 acquisitions/sec). Fluorescence signals were analyzed by software Absolute Quantification Analysis Using the Second Derivative Maximum Method (Roche). This analysis method used crossing point (Cp) values to extrapolate initial concentration of target DNA in each sample (Larionov et al. 2005, Roche instrument manual v1.5, pp. 167-169). Transgene copy number (EGFP gene) in each transgenic plant analyzed was determined based on comparison of Cp-extrapolated concentrations with in-run standard curve derived from EGFP gene-containing plasmid. Statistical analysis. Experimental data were analyzed using Proc GLM and ANOVA procedures of SAS software,

288 Dhekney et al. 2001 (SAS Institute, Cary, NC). Treatment means were compared using Student-Newman-Keuls test. Results Effect of callus induction medium. Production of transgenic callus and SE lines using DM (Li et al. 2001), GS1CA (Franks et al. 1998), or NB (Le Gall et al. 1994) callus induction media and procedures varied among Vitis genotypes. Transformed embryogenic cultures of Thompson Seedless and Seyval blanc produced the highest percentage of transgenic calli and SE lines on DM medium (Figure 1). While there was no difference in the percentage of transgenic calli produced from Cabernet franc, Merlot, and Superior Seedless on DM and GS1CA medium, the percentage of transgenic SE lines obtained in these genotypes was significantly greater on DM medium (Figure 1). As determined by GFP expression, cocultivated embryogenic cultures of Ramsey, Harmony, and Richter 110 produced a significantly greater percentage of stable transgenic calli on DM and GS1CA medium compared to NB medium. However, the calli was of a friable nonembryogenic type (Figure 2A, B). In contrast, compact transgenic callus was produced on NB medium, which resulted in production of transgenic SE lines after transfer to X6 medium (Figure 2C, D). Effect of embryogenic culture age. No significant difference was observed in transgenic callus formation between 4, 8, and 12 mo cultures (data not shown). However, culture age affected recovery of transgenic SE lines among varieties (Figure 3). No significant differences were observed in production of transgenic SE lines between 4, 8, and 12 mo cultures of Superior Seedless and Merlot, whereas 8 mo cultures of Thompson Seedless produced a greater percentage of SE lines compared to 4 and 12 mo cultures. A significant reduction in the percentage of transgenic lines obtained was observed in 8 and 12 mo transformed Seyval blanc cultures compared to 4 mo cultures, whereas Cabernet franc, Cabernet Sauvignon, and Shiraz failed to produce transgenic SE from 8 and 12 mo transformed cultures (Figure 3). Among the Vitis genotypes studied, the maximum percentage of transgenic SE lines were recovered from Thompson Seedless (36.0%) followed by Seyval blanc (23.0%) and Superior Seedless (22.5%). Effect of cotyledon excision on plant recovery. Plant recovery from germinated SE was genotype-dependent. Transgenic plants of V. vinifera and V. riparia were recovered after 3 weeks of culture on MS1B medium. Excision of cotyledons caused the faster development of robust shoots (Figure 4A) compared to controls (Figure 4B) and resulted in a better plant recovery in transgenic Cabernet franc, Cabernet Sauvignon, Orange Muscat, Superior Seedless, and V. riparia Gloire (Table 1). Transgenic plants of Ramsey, Harmony, and Richter 110 were recovered from germinated SE only after 9 weeks of culture and cotyledon excision resulted in reduced plant recovery compared to the control. Transgenic SE and plant lines obtained. Time required for recovery of transgenic SE from transgenic callus varied Figure 1 Effect of three callus induction media on production of transgenic callus and SE lines in Vitis. SE of V. vinifera Cabernet franc, Merlot, Superior Seedless, and Thompson Seedless, Vitis hybrids Harmony, Richter 110 and Seyval blanc, and V. champinii Ramsey at the mid-cotyledonary stage of development were transformed with Agrobacterium and transferred to Petri dishes containing callus induction medium. Bars represent average data from four replicated Petri dishes containing 30 SE explants. Statistical analysis carried out using Proc GLM and ANOVA procedures of SAS. Bars represented by the same letter were not significantly different according to Student-Newman- Keuls test (α = 0.05).

Genetic Transformation of Vitis 289 from 5 to 60 weeks according to genotype (Table 2). Transgenic plants were recovered from 11 V. vinifera genotypes, V. champinii, V. rupestris, V. riparia, and 5 Vitis hybrids. Molecular analysis of transgenic plants. The presence of the egfp transgene was confirmed by its amplification from genomic DNA of eight transgenic lines of different genotypes (Figure 5, lanes 1-8) and the positive control (lane P), whereas no amplification was obtained from genomic DNA of the non-transformed plant (lane 9). Transgene copy number was estimated in eight transgenic plant lines using quantitative real-time PCR. External controls (corresponding to 1 5 copies of plasmid DNA containing the egfp transgene) were used to establish a standard curve for evaluation of transgene copy number. Cp values extrapolated from plasmid DNA samples showed a high degree of proximity to the calculated value of plasmid DNA in each reaction (Table 3, sample 1 5). Extrapolated Cp values from transgenic plants revealed three plant lines with three copies, two plant lines with two and four copies, respectively, and one plant line with a single copy of the egfp transgene (Table 3, sample 6 13). Results from repeated experiments were identical. Melting curve analysis of real-time PCR products indicated a single peak fluorescence profile was produced by copy number controls and transgenic plant lines, which was significantly different from the fluorescence profile produced by the non-transformed plant (data not shown), thereby differentiating target specific amplification from nonspecific background signal noise. Figure 2 Transgenic callus and SE production on DM and NB medium in V. champinii Ramsey. Friable callus produced on DM medium (A) was transgenic (B) but nonembryogenic compared to compact callus produced on NB medium (C), which produced transgenic SE (D) after transfer to embryo development medium. Figure 4 Effect of cotyledon excision on transgenic plant recovery from germinated SE. Cotyledons of germinated SE were excised after 2 weeks of culture on MS1B medium. Shoot development was enhanced from germinated SE subjected to cotyledon excision (A) compared to the control (B), which failed to produce a shoot. Arrow indicates the area of cotyledon excision. Table 1 Effect of cotyledon excision on transgenic plant recovery in Vitis. Each treatment consisted of five SE in a Petri dish containing MS1B medium, replicated four times. Figure 3 Recovery of transgenic SE lines from 4, 8, and 12 month embryogenic cultures of Vitis. SE of V. vinifera genotypes at 4, 8, and 12 months after initiation were transformed with Agrobacterium and transferred to DMcck medium. The percentage of transgenic SE lines obtained was recorded after transfer to X6cck70 medium. Bars represent average data from four replicated Petri dishes each containing 30 SE. Statistical analysis carried out using Proc GLM and ANOVA procedures of SAS. Bars represented by the same letter were not significantly different according to Student-Newman-Keuls test (α = 0.05). Variety Intact cotyledon Plant recovery (%) a Excised cotyledon Recovery time (weeks) V. champinii Ramsey 33.3 a b 6.7 b 9 V. riparia Gloire 20.0 b 53.0 a 5 Vitis vinifera Cabernet franc 6.7 b 46.7 a 3 Cabernet Sauvignon 0.0 b 26.7 a 3 Orange Muscat 0.0 b 73.3 a 3 Superior Seedless 20.0 b 66.7 a 3 Vitis hybrids Harmony 40.0 a 0.0 b 9 Richter 110 73.3 a 20.0 b 9 a Values represent percentage of observations for four replications (20 SE total). b Means between columns represented by the same letter were not significantly different according to Student-Newman-Keuls test (α = 0.05).

290 Dhekney et al. Discussion We previously reported an optimized protocol for grapevine transformation (Li et al. 2006), which was further improved to reduce post-cocultivation culture necrosis and increase transgenic plant recovery (Li et al. 2008). In the current study, application of the improved protocol was extended to test other previously published procedures (Franks et al. 1998, Le Gall et al. 1994), and thus produce transgenic plants from a wide range of Vitis species and interspecific hybrids. SE at the mid-cotyledonary stage of development were used as explants for transformation with Agrobacterium. Earlier studies of grapevine transformation compared embryogenic callus (tissue type I) and SE (tissue type II) for cocultivation and reported the highest efficiency of transformed embryo recovery when embryogenic callus was used (Franks et al. 1998). In our studies, embryogenic callus was found to be unsuitable for cocultivation when compared to SE, as indicated by transient GFP expression and transgenic callus formation (data not shown). We have repeatedly demonstrated high transient and stable GFP expression levels when SE were used (Dhekney et al. 2008, Li et al. 2001, 2004, 2006, 2008). The production of SE on three callus induction media was genotype-dependent. Although transgenic callus was produced on all media, V. vinifera genotypes produced a higher number of transgenic SE when cultured on DM medium followed by transfer to X6 medium. This response could be attributed to a difference in the composition of these media and a propensity for greater embryogenic response on medium with high inorganic N. X6 medium differs significantly in its inorganic N source and levels (Li et al. 2001) from GS1CA (Franks et al. 1998). Growth and differentiation of embryogenic cultures is affected by source and ratio of NO 3 :NH 4 ions in the culture media (Lelijak-Levanic et al. 2004, Niedz 1994, Samoylov et al. 1998). Thus, a higher level of inorganic N and the presence of NH 4 Cl as a source of NH 4 ion in X6 medium may have contributed to a greater embryogenic response from transgenic calli. Ramsey and certain Vitis hybrids produced transgenic SE only when cultured on NB medium for callus induction followed by transfer to X6 medium for embryo development. This response may be attributed to the composition of NB medium (half-strength MS salts and lower growth regulator concentrations than DM and GS1CA), which resulted in the production of compact embryogenic callus compared to rapidly proliferating nonembryogenic callus produced on DM and GS1CA medium. Among the genotypes tested, Thompson Seedless, Superior Seedless, and Merlot produced transgenic SE lines irrespective of culture age. In contrast, no SE lines were recovered from 8 and 12 mo cultures of transformed Cabernet franc, Cabernet Sauvignon, and Shiraz. This response is attributed to differences in genotypes to maintain their embryogenic potential under long-term culture conditions. Grapevine somatic embryos proliferate by direct secondary embryogenesis with new embryos emerging from epidermal Variety Table 2 Summary of transgenic SE and plant lines recovered from Vitis. GFP expression (%) a SE lines (n) b Recovery time (weeks) c Plant lines recovered (n) b V. champinii Ramsey 84.00 37 16 23 V. riparia Gloire 79.00 8 16 4 V. rupestris St. George 28.13 72 6 4 V. vinifera Cabernet franc 72.20 39 56 19 Cabernet Sauvignon 59.60 4 24 1 Chardonnay 44.40 8 6 4 Merlot 68.80 374 8 160 Orange Muscat 19.60 4 60 4 Pinot noir 10.00 19 8 5 Sauvignon blanc 24.66 44 8 4 Shiraz 72.20 42 24 28 Superior Seedless 76.60 123 6 89 Thompson Seedless 81.10 1575 6 975 Zinfandel 52.20 5 12 1 Vitis hybrids Conquistador 68.80 20 6 5 Freedom 21.78 7 6 1 Harmony 68.90 34 18 14 Richter 110 71.20 18 18 4 Seyval blanc 67.80 477 6 201 a Transient GFP expression was recorded as the percentage of SE explants exhibiting GFP fluorescence vs the total number of cocultivated SE explants. An explant that exhibited 10 or more GFP foci was considered to be GFP positive for transient expression. b The number of transgenic SE and plant lines represents combined data from all transformation experiments. c The time required for transgenic SE recovery was recorded as the number of weeks required for development of a transgenic SE on embryo development medium after transformation with Agrobacterium. Figure 5 PCR analysis of genomic DNA isolated from leaves of transgenic and non-transformed plants of Vitis. A 717 bp DNA fragment corresponding to the egfp gene sequence was amplified from transgenic V. champinii Ramsey (lanes 1, 2), V. riparia Gloire (3, 4), V. vinifera Merlot (5, 6), Vitis hybrid Harmony (7, 8) and the positive control plasmid (lane P) used in transformation, but not from a non-transformed V. vinifera plant (9).

Genetic Transformation of Vitis 291 Table 3 Estimation of transgene copy number in transgenic plants using quantitative real-time PCR. No. Sample name Mean CP a STD CP b Mean concn c STD concn Est. copy no. 1 Plasmid- 1 copy 28.4340 0.1856 1.0207 0.2061 1 2 Plasmid- 2 copies 27.4456 0.1265 2.3875 0.2015 2 3 Plasmid- 3 copies 26.7996 0.0782 3.3739 0.1064 3 4 Plasmid- 4 copies 26.0703 0.2375 4.0887 0.1597 4 5 Plasmid- 5 copies 24.8307 0.0639 5.0001 0.0520 5 6 V. champinii Ramsey-T1 27.2822 0.0123 2.6478 0.0196 2 7 V. champinii Ramsey-T 2 25.7493 0.0590 4.3059 0.0415 4 8 V. riparia Gloire-T1 26.8239 0.0179 3.3421 0.0249 3 9 V. riparia Gloire-T2 26.9160 0.0580 3.2106 0.0842 3 10 Vitis hybrid Harmony-T1 27.6689 0.1151 2.0371 0.1775 2 11 Vitis hybrid Harmony-T2 28.0849 0.0517 1.4344 0.0687 1 12 V. vinifera Merlot-T1 26.6292 0.0837 3.5938 0.1010 3 13 V. vinifera Merlot-T2 25.9013 0.0477 4.1994 0.0326 4 14 V. vinifera control d 31.7282 0.0713 0.0033 0.0006 0 a Average values of crossing points (CP) from three sample replicates. b Standard deviation values for CP. c Average DNA concentration values extrapolated from CP values. d Sample values for non-transformed V. vinifera Thompson Seedless. or subepidermal cells (Gray 1995, Jayasankar et al. 2003). The time period for maintenance of embryogenic cultures varies with genotype. For instance, embryogenic cultures of Thompson Seedless could be maintained in culture for 48 months or longer (Gray 1995), whereas other genotypes lost their morphogenetic potential or differentiated completely into germinated embryos during the maintenance period (Coutos-Thevenot et al. 1992). Thus, the capacity for an embryogenic culture to produce embryos and plants after transformation appears to be at least partially dependent on the period of time it can be maintained without significant loss in regeneration potential. Transgenic plants of Vitis vinifera genotypes were obtained from germinated SE in 3 weeks and cotyledon excision increased the number of plants recovered, confirming our earlier results demonstrating the beneficial effects of cotyledon excision (Li et al. 2008). In contrast, development of germinated SE was slow in Ramsey and Vitis hybrids, where cotyledon excision resulted in a decline in the recovery of transgenic plants. The observed results were not due to transgene effects since similar results were obtained with non-transgenic germinated SE of Vitis genotypes (data not shown). The observations appear to be consistent with earlier reports of poor root development in Ramsey (Kose 2007). Thus, the effect of cotyledon excision on plant recovery is variety-dependent and must be determined experimentally for each variety. The maximum number of transgenic SE and plant lines were obtained from Thompson Seedless, followed by Seyval blanc, Merlot, and Superior Seedless. Molecular analysis of transgenic plants using PCR and quantitative real-time PCR demonstrated presence and the copy number of the egfp transgene. Plants with single as well as multiple transgene copy number were recovered. However no visual differences in GFP expression among plants with variable number of transgene insertions were observed in mature plant parts (data not shown). Transgenic vines exhibited normal vegetative growth and development compared to non-transgenic vines grown in the field. Our results contrast earlier reports where plants regenerated through somatic embryogenesis exhibited an uncharacteristic phenotype from the accepted ampelographic phenotype, suggesting that plants obtained from somatic embryos exhibited a juvenile phenotype similar to seedlings (Franks et al. 1998). Conclusion Transgenic plants from an increasing number of V. vinifera genotypes and rootstocks are now being routinely produced. The improved protocols described earlier together with current modifications can be extended to produce transgenic plants from additional Vitis species and genotypes. Results of this study demonstrate that obstacles to transformation have been overcome so that all molecular biology-based approaches to crop improvement now are possible for Vitis. Literature Cited Alleweldt, G., and J.V. Possingham. 1988. Progress in grapevine breeding. Theor. Appl. Genet. 75:669-673. Coutos-Thevenot, P., I. Goebel-Tourand, M.C. Mauro, J.P. Jouanneau, M. Boulay, A. Deloire, and J. Guern. 1992. Somatic embryogenesis from grapevine cells. I. Improvement of embryo development by changes in culture conditions. Plant Cell Tissue Org. Cult. 29:125-133. Das, D., M. Reddy, S. Upadhyaya, and S. Sopory. 2002. An efficient leaf disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.). Plant Cell Rep. 20:999-1005. Dhekney, S.A., Z.T. Li, M. Dutt, and D.J. Gray. 2008. Agrobacterium-mediated transformation of embryogenic cultures and

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